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J. Biol. Chem., Vol. 277, Issue 19, 16705-16711, May 10, 2002
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From the Department of Biological Sciences, University of Alberta,
Edmonton, Alberta T6G 2E9, Canada
TraM is a DNA binding protein required for
conjugative transfer of the self-transmissible IncF group of plasmids,
including F, R1, and R100. F TraM binds to three sites in F
oriT: two high affinity binding sites, sbmA and
sbmB, which are direct repeats of nearly identical sequence
involved in the autoregulation of the traM gene; and a
lower affinity site, sbmC, an inverted repeat important for
transfer, which is situated nearest to the nic site where
transfer originates. TraM bound cooperatively to its binding sites at
oriT; the presence of sbmA and sbmB
increased the affinity for sbmC 10-fold. Bending of
oriT DNA by TraM was minimal, suggesting that TraM, a
tetramer, was able to loop the DNA when bound to sbmA and
sbmB simultaneously. Hydroxyl radical footprinting of DNA
of sbmA and sbmC revealed that TraM contacted
the DNA within a region previously delineated by DNase I footprinting.
TraM protected the CT bases within the sequence CTAG, which occurred at
12-base intervals on the top and bottom strand of sbmA,
most consistently with other protected bases. The footprint on
sbmC revealed that the predicted inverted repeats were
protected by TraM with a pattern that began at the center of the
repeats and radiated outward at 11-12 base intervals toward the
5'-ends of either strand. At high protein concentrations, this pattern
extended beyond the footprint defined by DNase I, suggesting that the
DNA was wrapped around the protein forming a nucleosome-like structure,
which could aid in preparing the DNA for transfer.
Characterizing the DNA Contacts and Cooperative Binding of F
Plasmid TraM to Its Cognate Sites at oriT*
and
*
This work was supported in part by the Canadian Institutes
for Health Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by an Alberta Heritage Foundation for Medical Research
Studentship. Present address: the Laboratory of Biochemistry, NCI,
National Institutes of Health, Bldg. 37, Rm. 6044, 37 Convent Dr.,
Bethesda, MD 20892-4255.
§
To whom correspondence should be addressed: Dept. of Biological
Sciences, CW405 Biological Sciences Bldg., University of Alberta, Edmonton, Alberta T6G 2E9, Canada. Tel.: 780-492-5172; Fax:
780-492-9234; E-mail: laura.frost@ualberta.ca.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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